Project 1: Functional characterization of candidate cancer genes identified by genomic analysis of serous endometrial tumors During previous reporting periods, we published one of the first whole exome sequencing studies of serous endometrial tumors (Le Gallo et al., Nature Genetics 2012; 44:1310-5). The major finding of that study was our discovery that in serous endometrial tumors the FBXW7, CHD4, and SPOP genes are mutated at statistically significantly higher rates than the background mutation rate. This observation strongly suggests that somatic mutations in FBXW7, CHD4, and SPOP are pathogenic driver mutations that confer a selective advantage in serous endometrial tumorigenesis. However, proof of this hypothesis requires functional studies of the mutant proteins. Within the present reporting period we initiated functional studies to determine how mutations in FBXW7 and SPOP, which both encode the substrate recognition components of ubiquitin ligase complexes, affect their function. In ongoing and planned studies that will extend into the next reporting period, we are characterizing the function of FBXW7 and SPOP mutants, using both biochemical and cell-based approaches. We are focusing our efforts on recurrent hotspot mutations that are present in endometrial carcinomas, but are rare or absent in other types of cancer, and which we therefore hypothesize are likely to be pathogenic driver mutations that contribute to endometrial tumorigenesis. Project 2. Identification of somatic mutations that underlie clear cell endometrial cancer During the previous reporting periods, we subjected DNAs from clear cell endometrial tumors and from the matched normal tissues to whole exome sequencing at the NIH Intramural Sequencing Center. In the past reporting period we: 1. Analyzed the exome sequencing data by filtering germline variants (present in both tumor and normal DNAs) from somatic variants (present exclusively in the tumor DNA). 2. Subjected the somatic variants to orthogonal validation using Sanger sequencing to distinguish true somatic mutations from false-positive calls. 3. Prioritized a subset of nonsynonymously, somatically mutated genes, which we have deemed candidate cancer genes, for further analysis in a mutation prevalence screen. The mutation prevalence screen was initiated during the past reporting period and extended into the current reporting period. In the current reporting period we: 1. Completed the first stage of the mutation prevalence screen by sequencing our series of candidate cancer genes from another large cohort of clear cell ECs provided by extramural and international collaborators. This led us to identify potential somatic mutations, which we subsequently subjected to confirmation by sequencing both the tumor DNA and matched normal DNA. 2. Started the second phase of the mutation prevalence screen in which we identify, sequence, and reanalyze exons that had poor initial sequence coverage. In the incoming reporting period we will: 1. Finish sequencing to attain high-level coverage or targeted genomic regions. 2. Calculate the mutation rate of the individual genes in clear cell endometrial cancer. If a gene is mutated at a significantly higher rate than the background mutation rate (which we will empirically determine) this provides strong genetic evidence that the mutated gene is likely to be a pathogenic driver gene that confers a selective advantage in clear cell endometrial tumorigenesis. Should these studies reveal a novel, significantly mutated gene, we plan to initiate a limited number of biochemical and cell-based studies to determine the functional consequences of the observed mutations. 3. Prepare a manuscript describing our findings. Project 3. Interrogation of somatic copy number alterations in serous and clear cell endometrial cancer In parallel to the mutational analyses described in Project 1 and 2, we are searching for somatic copy number alterations in serous and clear cell endometrial tumor genomes. In previous reporting periods, we used high-density SNP arrays to catalogue somatic genomic copy number alterations (gains and losses) that occur in serous and clear cell endometrial tumors. In the past reporting period, we used an independent method to search for copy number alterations in these tumors. Specifically, in collaboration with the Mullikin lab (NHGRI), we have used the ExomeCNV algorithm to identify copy number alterations within the short sequence reads we generated for serous and clear cell endometrial tumor exomes (Project 1, and Project 2 respectively). In the current reporting period and in ongoing studies that will extend into the next reporting period, we will compare the SNP array and ExomeCNV data to identity concordant regions of copy number gain or loss identified by these two independent methods. In ongoing work, initiated within the current reporting period, we are preparing a manuscript describing some of our findings. Collaborative projects Tumorigenesis is driven by genomic and epigenomic alterations, including altered methylation states. In the past, current and incoming reporting periods, we are collaborating with Dr. Laura Elnitski (NHGRI/NIH) to determine the genome-wide methylation states of endometrial carcinomas, including the serous and clear cell subtypes.